The application of genetic engineering techniques for the induction of bone formation currently being evaluated in pre-clinical and clinical studies for the treatment of numerous pathologic disorders, including osteoporosis and nonhealing long bone fractures. Bone repair and reconstruction also has significant implications for improving the healing of facial fractures, the restoration of bony tissues in developmental craniofacial disorders, and the regeneration of alveolar bone in periodontal disease. The induction of osteogenesis throughout the skeletal system using bone morphogenetic proteins (BMPs), typically on a biologic carrier, has had success in the experimental setting, and is now being tested for clinical use. Our laboratory has recently demonstrated that direct BMP gene transfer techniques can also be utilized to stimulate bone formation in ectopic and orthotopic regions, including critical size mandibular defects. However, first generation BMP adenoviral vectors currently in use are limited by the immune responses they elicit in immunocompetent animals. The engineering of novel BMP viral vectors, however, should advance the clinical efficacy of this unique technique for the treatment disorders involving bone loss. We hypothesize in this grant proposal that the efficacy of BMP genetic therapy can be enhanced by: 1) the generation of novel viral vectors which improve transgene expression, 2) the use of tissue-specific control elements to localize BMP expression to specific target cells, 3) the use of an inducible expression system to temporally regulate BMP production within target cells, and 4) the utilization of ex vivo gene therapy techniques. To test these hypotheses, we propose the following studies: I. To decrease the antigenicity of the first generation adenoviral vectors and increase the length of transgene expression, we will construct, produce, and test two novel BMP vectors, including a gutless adenoviral vector and an adeno-associated viral vector. II. To restrict BMP expression to specific cell populations, we will produce and test BMP viral vectors with the transgene under control of the osteocalcin promoter (osteoblast specific). III. To externally regulate osteogenesis, we will produce and test a BMP viral vector with the transgene under control of tetracycline responsive control elements. IV. To evaluate ex vivo gene therapy, human mesenchymal stem cells will be transduced with BMP genes and delivered on a Type I collagen scaffold. The osteogenic activity of these novel approaches will be evaluated in a rigorous fashion using immunohistochemistry, histology, and computed tomography in heterotopic and orthotopic locations, providing the scientific foundation for future basic and clinical BMP gene therapy studies.